Toxoplasma gondii is an important opportunistic pathogen that causes severe complications in immuno compromised individuals and can be the cause of congenital defects when a fetus is infected during the first trimester of gestation. Toxoplasma, like the other member of the phylum Apicomplexa, such as Plasmodium and Cryptosporidium, is an oblegate intracellular parasite that actively invades its animals host cells,a process unique to this calls of parasites. Although host cell invasion is the most important step in the life cycles of this class of parasites, little is known about its underlying mechanism and regulation. AN understanding of the molecular mechanism of the invasion process in T. gondii could potentially identify targets for chemotherapeutic intervention, and could also serve as a model for understanding this process in other apicomplexan parasites. We have found that invasion of Toxoplasma into its host cells is regulated by the protein kinase C (PKC) pathway. A number of inhibitors, specific for different players in this pathway, inhibit the invasion process after attachment and reorientation, but prior to the actual start of invasion. This pathway does not appear to be required at any other step in the Toxoplasma tachyzoite life cycle. We have tentatively identified a parasite PKC (TgPKC) and four PKC substrates, the downstream effectors. This project has two major specific aims. The first is to characterize the PKC enzyme (s) of T. gondii and analyze its sensitivity to a range of inhibitors and activators. We will also attempt to generate novel TgPKC-specific peptide inhibitors. Because it is unlikely that sufficient quantities of PKC can be purified from Toxoplasma tachyzoites for a thorough enzymatic analysis and extensive search for effective and specific inhibitors or activators, the initial focus will be on the molecular cloning of cDNAs and genes encoding TgPKC and their high level expression in the baculovirus expression system. The second specific aim is to characterize the PKC substrates in Toxoplasma. We have identified four potential PKC substrates and will attempt to purify these for further characterization with an approach used successfully in the purification of several major PKC from mammalian cells. Further characterization will include the generation of monospecific antibodies to be used for the molecular cloning of sequences encoding these proteins and for immunolocalization of these proteins in parasites. Depending on the information thus obtained, the TgPKC substrates will be further characterized.